DE3923895A1 - METHOD FOR SEQUENCING DESOXYRIBONUCLEIC ACIDS - Google Patents

Abstract

The method comprises subjecting the deoxyribonucleic acid in a concentration of 10<-16> to 2 x 10<-14> mol per mu l, in the case of double-stranded deoxynucleic acid which is not in the supercoiled form preferably in a concentration of 10<-16> to 3 x 10<-15> mol per mu l, a) to a heat-denaturation step b) to an annealing step and c) to a nucleic acid synthesis step with partial chain termination, repeating steps a) to c) 10 to 60 times and subsequently fractionating the resulting deoxynucleic acid fragment mixture according to length, and determining the sequence from the fragment spectrum. o

Description

The analysis of the nucleotide sequence of nucleic acids is of central importance Technology in molecular biology.

There are currently two basic techniques for routine sequences adornment of DNA. On the one hand, it is about Maxam and Gilbert's method. (Methods Enzymol. 65 (1980), 499-560) and secondly the Sanger method (Proc. Natl. Acad. Sci. USA 74 (1977), 5463).

Sanger sequences are almost exclusively used for DNA sequencing ornament used, starting from an oligonucleotide primer, its sequence to a part of the DNA strand to be sequenced is complementary in the presence of a mixture of the four deoxy nucleotide triphosphates and a dideoxynucleotide triphosphate of one Complementary copies of the strand to be sequenced are produced by polymerase will. The decisive factor here is the use of dideoxynucleotide tri phosphate, because after their incorporation into the newly synthesized DNA chain extension is no longer possible if there is no hydroxyl group and sequence-specific chain terminations are generated in this way to let. The chain extension reactions are usually separated approaches for all four possible mixtures of the four deoxy nucleotide triphosphates and a dideoxynucleotide triphosphate leads. The length of the generated ones is then separated Nucleic acid fragments using conventional electrophoretic methods enable direct reading of the nucleotide sequence. To differentiate the newly synthesized DNA from the DNA to be sequenced are either the Oligonucleotide primer, at least one of the four deoxynucleotides or the Dideoxynucleotides with e.g. Radioisotopes or more recently Fluorescent dyes marked.

The reactions are carried out for all currently used Two-to three-stage procedure: In a first (for single-stranded DNA unnecessary) step will be a separation of the two complementary Strands of DNA to be sequenced using elevated pH or heat taken. This is followed at the beginning of the so-called annealing reaction with an increased Temperature (about 65 ° C) the addition of the oligonucleotide primer, which is then in The course of the cooling of the solution binds to the strand to be sequenced. The third step is for one for the polymerase used suitable temperature the actual sequencing reaction.  

At the moment there are practically only three different types of poly merases used for DNA sequencing: the Klenow fragment of the E. Coli polymerase I, modified forms of T7 polymerase and the heat stable Taq polymerase from Thermus aquaticus.

With the currently available methods, all single-stranded DNA samples as well as double-stranded DNA in supercoil form Samples are routinely sequenced provided the latter one critical size (about 20 kilobases). One in the Routine proven regulation for the sequencing of supercoil DNA with However, Taq polymerase does not currently exist. That would be especially desirable because the higher possible Reaction temperature problems caused by the formation of secondary structure structures are caused, eliminated.

In addition to the restrictions listed above, especially the Sequen prepares ornamentation of linear double-stranded DNA molecules problems. Such DNA molecules occur in particular as end products of the so-called Polymerase chain reaction (PCR) (see e.g. Mullis et al., Methods Enzymol. 155 (1987), 1973-1977). In addition to wide applicability this is a highly sensitive method in molecular biological research for the in vitro amplification of DNA fragments, especially for analysis the molecular basis of hereditary diseases, in forensic identi fication problems and in the prenatal diagnosis of very large Significance, since in all the cases mentioned only small amounts of nuclein acid are available for sequence analysis according to the usual Procedures are not sufficient.

A sequencing method for PCR fragments based on the method of Sanger, was developed by Saiki et al. (Science 239 (1988), 487-491) described. However, this method is to a large extent one optimal execution of the individual successive reaction steps depending on what complicates their applicability in routine operation. Furthermore processes based on the Maxam-Gilbert principle have also been described build a chemical strand break (e.g. Keohavong et al., DNA 7 (1) (1988) 63-70; Voss et al., Nucl. Acids, Res. 17 (7) (1989), 2517-2527). The disadvantage of this method is the need that after the PCR complex chemical reactions with partly toxic substances must be carried out. In addition, was over a very elegant process reported in principle, in which already during the PCR involves a sequence-specific incorporation of thio-deoxynucleotides, which were then defined using an alkylation reaction Strand breaks are generated (Nakamaye et al., Nucl. Acids Res. 16 (21) (1988), 9947-9959). However, this procedure has the decisive one  Disadvantage that only low signal intensities can be generated and the area accessible to sequencing on a maximum of 250 bases is limited.

In addition, methods have recently been reported for which the Taq polymerase is used and the usual reaction cycle a few times (Levedakou et al., Bio Techniques 7 (1989) 438-422; Carothers et al. (1989) BioTechniques 7: 494-499).

When using fluorescent dyes to mark the new synthesized fragments, it is extremely desirable to have a method for To have available with a much higher yield of fragments can be obtained than is possible with the conventional methods.

The invention relates to a method for sequencing deoxy ribonucleic acids, which consists in that the deoxyribonuclein acid

• a) a heat denaturation step,
• b) an annealing step and
• c) a nucleic acid synthesis step with partial chain termination

subjects steps a) to c) repeated 10 to 60 times and then the resulting deoxynucleic acid fragment mixture of length after separating and determining the sequence from the fragment spectrum.

The heat denaturation step takes place at temperatures between 92 and 98 ° C, preferably 96 ° C.

The annealing step works well at temperatures between 35 and 75 ° C.

The nucleic acid synthesis step is based on an oligonucleotide primer from, which is complementary to a section of the individual to be sequenced must be strangs. This primer is about 10 to 50, preferably 15 to Is 30 nucleotides long, is attached to the DNA to be analyzed. The molar ratio between oligonucleotide primer and to be sequenced Sequence is preferably between 2 and 30, i.e. there will be too per mole sequencing sequence between 2 and 30 moles of primer used. After that the primer is in the presence of a mixture of the four deoxy nucleotide triphosphates and at least one dideoxynucleotide triphosphate extended to complement the DNA strand to be sequenced. The extensions tion is carried out enzymatically with a heat-stable polymerase, e.g. with the Taq polymerase. The nucleic acid synthesis step succeeds in the case of Taq polymerase works well at a temperature of 65 to 85 ° C.  

Steps a) to c) are about 10 to 60 times, preferably 10 to Performed 30 times in a row.

The addition of dideoxynucleotide triphosphates serves to synthesize the abort complementary DNA sequence-specifically. Can break the chain one can use a particular one of the four dideoxynucleotide triphosphates. In In this case, DNA synthesis must be performed 4 times, i.e. with every dideoxynucleotide triphosphate once.

However, a mixture of the 4 dideoxynucleotide triphosphates can also be used turn. In this case, it is sufficient to carry out the synthesis only once.

In order to recognize the newly synthesized nucleic acids, they have to be marked. This can be done by using labeled oligonucleotide primers or from labeled deoxy or dideoxy nucleotide triphosphates. The label can be labeled with a radioisotope, an enzyme, a specific Binding site for a detectable molecule or preferably with a Fluorescent dye done.

The implementation of the individual steps mentioned above is known (e.g. Tabor et al., Proc. Natl. Acad. Sci. USA 84: 4767-4771 (1987).

The method according to the invention has an advantage over those currently available DNA sequencing methods have the following advantages:

• a) It allows you to use PCR products in a reliable and simple manner be sequenced (Example 1).
• b) The method enables the sequencing of high-molecular, double-stranded DNA samples such as of lambda DNA (example 2) and generally the sequencing of double-stranded DNA, even if this is not in the supercoil form (Example 3).
• c) It is also applicable to single-stranded DNA and works with everyone DNA samples compared to conventional methods to a multiple higher yield of reaction products, so that a corresponding smaller amount of DNA is sufficient for sequencing and especially the applicability of the fluorescent label no longer is limited by the amount of DNA available.

The decisive difference between the method and The currently used methods of DNA sequencing according to Sanger are in the cyclical conduct of the sequencing reaction, being a very large one Number of cycles is used. The very large number of cycles  enables on the one hand when using a large excess of oligo nucleotide primer reaching comparatively very high temperature exploit. On the other hand, it leads to the extensive suppression of so-called. unspecific termination even in the case of problematic DNA samples or of non-optimized reaction conditions.

Example 1 Sequencing a PCR product PCR

0.1 fmol of a pUC18 derivative containing a 600 bp insert in the multi-cloning site was in the presence of 4 pmol of the (-21) standard primer (5′-CGT TGT AAA ACG ACG GCC AGT- 3 ′) and the reverse primer ( 5 ′ -CAC ACA GGA AAC AGC TAT GAC-3 ′) and in the presence of 20 nmol of the four deoxynucleotide triphosphates in a volume of 100 μl PCR buffer (50 mM KCl, 10 mM Tris -HCl, 1.5 mM MgCl ₂ , 0.01% gelatin, pH 8.3) amplified with 5 units Taq polymerase (Cetus).

For this purpose, the reaction mixture was overlaid with 100 ul mineral oil and in a programmable thermostat block (Programmable Dri-Block PHC-1, Techne) 30 temperature cycles consisting of 1.0 min. 96 ° C, 0.5 min. 37 ° C and 1.0 min. Subject to 72 ° C.

It was then carried out using a Biospin P30 column (biorad) according to the manufacturer's instructions and after equilibration of the column with the PCR buffers for the aqueous phase of the reaction solution Deoxynucleotide triphosphates.

Sequencing

The eluate from the Biospin column became a sequence without further treatment tion used. For the four sequencing reactions, the following were: Approaches used.

Each of the four reaction batches was 40 cycles analogous to the PCR reaction consisting of 1.0 min. 96 ° C, 0.5 min. 55 ° C and 1.0 min. Subject to 72 ° C.

The aqueous phases of the four reaction batches were then passed in succession other pipetted onto 20 µl 3 M NaOAc (pH 7.8). To this solution then 500 .mu.l of ethanol tempered to -20 ° C, the solution was mixed, 15 min. placed on dry ice and 15 min., at 13,000 rpm and RT centrifuged in an Eppendorf centrifuge 5415 C. That again with 800 ul 80% ethanol washed pellet was then 2 min under vacuum dried, in 2 µl 50 mM EDTA (pH 7.5) and 6 µl deionized formamide recorded, heated to 95 ° C for 2 min, cooled in ice water and in one DNA sequencer 370A (Applied Biosystems) electrophoresis (PAG with 6%  Acrylamide (AA) AA / bis-AA: 40: 2; Buffer: 0.089 mM Tris, 0.089 mM Borate, 2mM EDTA 7M urea pH 8.3; Power: 20 W) subjected. The Control of the sequencer and evaluation of the electropherograms was carried out with the software version 1.30 of the manufacturer.

The result obtained is shown in Fig. 1. A comparison between the determined sequence and the previously known sequence showed that apart from two problems, the described method can be used to uniquely determine at least 400 bases.

Example 2 Sequencing of lambda DNA

The sequencing of a lambda DNA (Lambda ZAP, Stratagene) was analogous Example 1 performed. It was just in place of the PCR product appropriate amounts of lambda DNA used, all other parameters remained unchanged.

The result is shown in Fig. 2. In this case too, the method described allowed the reliable determination of at least 400 bases.

Example 3 Sequencing of a linearized plasmid

The plasmid pUC18 was linearized with the restriction endonuclease XmnI and sequenced analogously to Example 1. Differing from example 1 were however 0.1 pmole (A and C reaction) or 0.2 pmole (G and T reaction) the DNA and 2 pmoles (A, C) or 4 pmoles (G, T) of the primers, and only ten temperature cycles of the type as in the PCR in Example 1 used.

The result is shown in Fig. 3. Apart from three unassignable bases, this experiment also made it possible to reliably determine at least 400 bases.

Claims (1)

Process for sequencing deoxyribonucleic acids, which consists in that the deoxyribonucleic acid

• a) a heat denaturation step,
• b) an annealing step and
• c) is subjected to a nucleic acid synthesis step with partial chain termination, steps a) to c) are repeated 10 to 60 times and then the resulting deoxynucleic acid fragment mixture is separated lengthwise and the sequence is determined from the fragment spectrum.